Not Applicable
This invention relates to the hormonally active, natural metabolite 1xcex1,24(S)-dihydroxyvitamin D2 and to methods of preparing this metabolite and the nonbiological epimer 1xcex1,24(R)-dihydroxyvitamin D2. This invention also relates to a pharmaceutical composition which includes a pharmaceutically effective amount of 1xcex1,24(S)-dihydroxyvitamin D2, to a method of controlling abnormal calcium metabolism by administering a pharmaceutically effective amount of the compound, and to a method of treating hyperproliferative diseases by administering the compound.
Vitamin D and its active metabolites are known to be important in regulating calcium metabolism in animals and humans. The naturally occurring form of vitamin D in animals and humans is vitamin D3. It has been shown that in animals, including humans, vitamin D3 is activated by being hydroxylated in the C25 position in the liver, followed by 1xcex1-hydroxylation in the kidney to produce the hormone 1xcex1,25-dihydroxyvitamin D3 [xe2x80x9c1xcex1,25-(OH)2D3xe2x80x9d]. See, U.S. Pat. No. 3,880,894. The major physiological pathway for catabolism of the vitamin D3 metabolites, 25-hydroxyvitamin D3 and 1xcex1,25-(OH)2D3, is initiated by C24-oxidation. Holick, M. F., Kleiner-Bossallier, A., Schnoes, H. K., Kasten, P. M., Boyle, I. T., and DeLuca, H. F., J. Biol. Chem., 248, 6691-6696 (1973).
Vitamin D2, on the other hand, is the major, naturally occurring form of vitamin D found in plants. Vitamin D2 differs structurally from vitamin D3 in that vitamin D2 has a methyl group at C24 and has a double bond between C22 and C23.
Shortly after their discovery, it seemed apparent that vitamin D3 and vitamin D2 had similar, if not equivalent, biological activity. It has also been commonly believed that the metabolism (i.e., the activation and catabolism) of vitamin D2 was the same as for vitamin D3. See, Harrison""s Principles of Internal Medicine: Part Seven, xe2x80x9cDisorders of Bone and Mineral Metabolism: Chap. 35,xe2x80x9d in E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. D. Wilson, J. B. Martin and H. S. Fauci (eds.), Calcium, Phosphorus and Bone Metabolism: Calcium Regulating Hormones, McGraw-Hill, New York, pp. 1860-1865. In this regard, the active form of vitamin D2 is believed to be 1xcex1,25-dihydroxyvitamin D2 [xe2x80x9c1xcex1,25-(OH)2D2xe2x80x9d]. Further, 24-hydroxy derivatives of 25-hydroxyvitamin D2 and 1xcex1,25-(OH)2D2, i.e., 24,25-dihydroxyvitamin D2 and 1xcex1,24,25-trihydroxyvitamin D2, are known, suggesting that catabolism of vitamin D2, like vitamin D3, proceeds through the same C24 oxidation step. Jones, G., Rosenthal, D., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Robinavich, D. and Shakked, Z., Biochemistry, 18:1094-1101 (1979).
It has recently been found, however, that an active analogue of vitamin D2, 1xcex1-hydroxyvitamin D2 [xe2x80x9c1xcex1-(OH)D2xe2x80x9d] has pharmacological properties distinctly different than those exhibited by its vitamin D3 counterpart, 1xcex1-hydroxyvitamin D3 [xe2x80x9c1xcex1-(OH)D3xe2x80x9d]. U.S. Pat. No. 5,104,864 discloses that 1xcex1-(OH)D2 will reverse the loss of bone mass in human osteoporotic patients when administered at dosages of 2.0 xcexcg/day or higher. Because of toxicity, dosage levels of 2.0 xcexcg/day or greater are not safely obtained with 1xcex1-(OH)D3.
Such distinct pharmacological properties may be explained fully, or in part, by the present inventors"" discovery that pharmacological dosages of 1xcex1-(OH)D2 administered to humans are metabolized in part to biologically active 1xcex1,24(S)-dihydroxyvitamin D2 [xe2x80x9c1xcex1,24(S)-(OH)2D2xe2x80x9d]. As explained in more detail below, the hydroxylation at the carbon-24 position of the 1-hydroxylated vitamin D2 molecule, represents an activation pathway peculiar to the vitamin D2 molecule.
While 1xcex1,24(S)-dihydroxyvitamin D3 and 1xcex1,24(R)-dihydroxyvitamin D3 [xe2x80x9c1xcex1,24(R/S)-(OH)2D3xe2x80x9d] have been chemically synthesized (U.S. Pat. No. 4,022,891) it has not been demonstrated that either is a natural compound found in biological systems. Furthermore, the present inventors have discovered that 1xcex1,24(S)-(OH)2D2 has distinctly different biological activity from that exhibited by 1xcex1,24(R/S)-(OH)2D3. For example, Ishizuka et al. have found that 1xcex1,24(R)-(OH)2D3 binds the 1,25-(OH)2D3 receptor site more tightly than does 1,25-(OH)2D3 itself. Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids, 37:1,33-42 (1981); Ishizuka, S., Bannai, K., Naruchi, T. and Hashimoto, Y., Steroids, 39:1,53-62 (1982). Using a similar assay, the present inventors have discovered that the 1xcex1,24(S)-(OH)2D2 is two-fold less competitive in binding the 1,25-(OH)2D3 receptor site than is 1,25-(OH)2D3. The present inventors have also found that 1xcex1,24(S)-(OH)2D2 shows a relatively poor binding affinity for the vitamin D serum binding protein which is evidence of a rather short half life indicative of low toxicity.
The present inventors have demonstrated the presence of circulating 1xcex1,24(S)-(OH)2D2 in humans administered 1xcex1-(OH)D2. This indicates that in animals and man, vitamin D2 is naturally metabolized to both 1xcex1,25-(OH)2D2 and 1xcex1,24(S)-(OH)2D2. The relative ratios of the two vitamin D2 hormones appear to vary according to the precursor and the amount of precursor presented to the C24 pathway. Thus, it appears that as dosages of 1xcex1-(OH)D2 are increased, the ratio of 1xcex1,24(S)-(OH)2D2 to 1xcex1,25-(OH)2D2 increases.
These results which are presented in more detail below, indicate that 1xcex1,24(S)-(OH)2D2 has the desirable characteristic of high biological activity with low toxicity. The fact that 1xcex1,24(S)-(OH)2D2 is a significant metabolite when pharmacological levels of 1xcex1-(OH)D2 are administered indicates that 1xcex1,24(S)-(OH)2D2 may be mediating the desirable pharmacological effects of 1xcex1-(OH)D2 and is a useful therapeutic drug for treating various types of disorders involving calcium metabolism.
Extensive research during the past two decades has also established important biologic roles for vitamin D apart from its classic role in bone and mineral metabolism. Specific nuclear receptors for 1xcex1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, are present in cells from diverse organs not involved in calcium homeostasis. For example, specific, biologically active vitamin D receptors have been demonstrated in the human prostatic carcinoma cell line, LNCaP, (Miller et al., 52 Cancer Res. (1992) 515-520). Vitamin D receptors have also been described for many other neoplastic cells, e.g., carcinomas of the breast and of the colon.
It has been demonstrated that certain vitamin D compounds and analogues are potent antiproliferative and prodifferentiative agents. For example, U.S. Pat. No. 4,391,802 issued to Suda et al. discloses that 1xcex1-hydroxyvitamin D compounds, specifically 1xcex1,25-dihydroxyvitamin D3 and 1xcex1-hydroxyvitamin D3, possess potent antileukemic activity by virtue of inducing the differentiation of malignant cells (specifically leukemia cells) to nonmalignant macrophages (monocytes), and are useful in the treatment of leukemia. Antiproliferative and differentiating actions of 1xcex1,25-dihydroxyvitamin D3 and other vitamin D3 analogues have also been reported with respect to prostate cancer cell lines. More recently, an association between vitamin D receptor gene polymorphism and prostate cancer risk has been reported, suggesting that vitamin D receptors may have a role in the development, and possible treatment, of prostate cancer.
These previous studies have focused exclusively on vitamin D3 compounds. Even though these compounds may be highly effective in promoting differentiation in malignant cells in culture, their practical use in differentiation therapy as anticancer agents is severely limited because of their equally high potency as agents affecting calcium metabolism. At the levels required in vivo for effective use as, for example, as antileukemic agents, these same compounds can induce markedly elevated and potentially dangerous blood calcium levels by virtue of their inherent calcemic activity. That is, the therapeutic use of 1xcex1,25-dihydroxyvitamin D3 and other vitamin D3 analogues as anticancer agents is precluded, or severely limited, by their side effects which include hypercalcemia and hypercalciuria. This indicates a need for compounds with greater specific activity and selectivity of action, i.e., vitamin D compounds with antiproliferative and prodifferentiating effects but which have low calcemic activity. Such compounds are xe2x80x9chypocalcemicxe2x80x9d vitamin D compounds. The need for such compounds is no greater than in the treatment of neoplastic and hyperproliferative diseases.
The present invention provides synthetic 1xcex1,24(S)-dihydroxyvitamin D2 [1xcex1,24(S)-(OH)2D2] which is a biologically-produced active form of vitamin D2. The biological form may also be referred to as 1xcex1,24(S)-dihydroxy ergocalciferol and is represented by the structure given hereinafter. The biological form of the compound has potent biological activity and rapid systemic clearance, indicating low toxicity.
The invention also encompasses a novel method of producing 1xcex1,24(S)-dihydroxyvitamin D2 which entails using ergosterol as a starting material, forming 24-hydroxyvitamin D2 and then, 1xcex1-hydroxlyating the 24-hydroxy compounds and separating the 1xcex1,24(S)-dihydroxyvitamin D2 epimer from the 1xcex1,24(R)-dihydroxyvitamin D2 epimer. In the course of this synthesis, novel intermediates are also produced. The crystalline form of 1xcex1,24(S)-dihydroxyvitamin D2 has further been found to have surprising stability and better biological activity than a white powder form of the compound.
The compound of the invention is useful in the treatment of various diseases characterized by vitamin D deficiency and various bone depletive disorders, in particular, treatment without the concomitant incidence of hypercalcemia or hypercalciuria. The compound of the invention is advantageously used as an active ingredient of pharmaceutical compositions for vitamin D deficiency diseases, for reversing or preventing the loss of bone mass or bone mineral content in persons predisposed to developing such loss, and for stabilizing bone density in persons suffering from renal osteodystrophy.
The compound of the invention is also useful as a topical and oral agent for treatment of certain skin disorders. The compound of the invention is advantageously used as an active ingredient in e.g., topical compositions which may also include other agents capable of ameloriating skin disorders.
The compound of the invention is also beneficial as a antiproliferative and prodiffentiative agent in the treatment of cancers and other hyperproliferative diseases.
Other advantages and a better appreciation of the specific adaptations, compositional variations, and physical and chemical attributes of the present invention will be gained upon an examination of the following detailed description of the invention, taken in conjunction with the accompanying drawings.